Large scale mixotrophic production systems

ABSTRACT

Systems for culturing mixotrophic microorganisms on a large scale in thermally stable open systems are disclosed. Embodiments of the system include features such as lit portions, dark portions, organic carbon delivery systems, gas delivery systems, submersible thrusters for mixing, and turning vanes for guiding fluid flow. Multi-functional embodiments of the turning vane provide guidance for fluid flow and other functions such as heat exchange, nutrient delivery, gas delivery, organic carbon delivery, delivery of light, and parameter measurement by sensors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/822,716, filed Aug. 10, 2015 and entitled Large Scale MixotrophicProduction Systems, which application is a continuation ofPCT/US2014/028604 filed Mar. 14, 2014 and entitled Large ScaleMixotrophic Production Systems, which claims the benefit of U.S.Provisional Application No. 61/798,969, filed Mar. 15, 2013, entitledMixotrophy Systems and Methods, and U.S. Provisional Application No.61/919,008, field Dec. 20, 2013, entitled Large Scale MixotrophicProduction Systems, the entire contents of which are hereby incorporatedby reference herein.

BACKGROUND

The large scale culturing of microorganisms has been establishedprimarily in two categories, phototrophy and heterotrophy. Phototrophycomprises culture conditions in which the microorganisms utilize lightas an energy source and inorganic carbon (e.g., carbon dioxide,bi-carbonate) for photosynthetic activity, which facilitates growth ofthe microorganism and the production of oxygen. Heterotrophy comprisesculture conditions in which microorganisms utilize organic carbon as anenergy and carbon source to facilitate growth of the microorganism andproduction of carbon dioxide. Phototrophy performed at a large scale inopen ponds is conventionally performed in non-axenic conditions, butlarge scale heterotrophy is conventionally performed in the axenicconditions of closed industrial fermenters.

A third category of microorganism culturing known as mixotrophy may alsobe used when the microorganism has the capability to use both light andorganic carbon as the energy source, and organic and inorganic carbon asthe carbon source. Mixotrophy may be performed in closed axenicconditions, but may also be performed in lower cost open non-axenicconditions. Mixotrophy provides the potential for an increased growthrate compared to phototrophic cultures and reduced capital costscompared to heterotrophic cultures. Additionally, the use of light inmixotrophy allows for a more diverse product profile to be produced(e.g., pigments, carotenoids) than may be produced in heterotrophiccultures which do not receive any light.

In the prior art, mixotrophy has largely been performed at laboratory orbench top scale, and not in large scale commercial production settings.The laboratory scale uses simple bioreactor systems, such as flasks andbubble columns, at small experimental culture volumes. The informationcoming out of such laboratory scale bioreactors is limited in itsusefulness to industry, as a flask cannot be easily scaled up to producecommercial quantities of microorganisms. Additionally, at the smallculture volumes of laboratory and bench top scale it is very easy tocontrol the culture conditions, such as pH, temperature, dissolved gascontent, and contamination. As the volume increases to commercial scale,the bioreactor system and microorganism culture faces new challenges notpresent at the laboratory scale.

The existing large scale production systems for phototrophic andheterotrophic cultures may be used for mixotrophy, but do not take fulladvantage of the mixotrophic culture conditions to produce an optimalyield or to culture large volumes of mixotrophic microorganisms in themost efficient manner. Large scale ponds used for phototrophic culturesare limited to shallow culture depths to allow light to penetrate theaqueous culture and be available to the microorganisms. Such shallowculture depths result in an inefficient use of land as the volume tosurface area ratio is low, and thus the yield of microorganisms andyield of microorganism produced products per surface area of land arenot optimized. Large scale fermenters used for heterotrophic culturingrequire high capital costs due to the materials needed to make the largesealed vessels, and mechanical mixing to properly distribute the gasesand organic carbon. The high capital cost of sealed fermenters reducesthe margins for profit, and the use of mechanical mixing may produceharmful shear stress for some species of microorganisms. Wastewatertreatment systems using microalgae in various stages of treatmentutilize large open ponds with a typical depth between 1 and 10 meters,but the systems are designed for optimization of water treatment and notmicroorganism production, and thus do not facilitate high culturedensities which results in low production yields of microorganisms.

To capitalize on the production ability and versatile product profile ofmixotrophic microorganisms, a high volume large scale production systemspecific to the mixotrophic culture methods and conditions is neededthat improves on existing deficiencies of large scale phototrophic,heterotrophic, and wastewater systems, and addresses the challenges notfaced in bench top scale mixotrophic cultures. Therefore, there is aneed in the art for a large scale mixotrophic production system for theefficient production of large volumes of mixotrophic microorganisms.

SUMMARY

Described herein are systems for culturing mixotrophic microorganisms ona large scale. Also described are multi-functional embodiments of aturning vane to provide guidance for fluid flow and other functions suchas heat exchange, nutrient delivery, gas delivery, organic carbondelivery, delivery of light, and parameter measurement by sensors.

In one embodiment of the invention, a mixotrophic bioreactor systemcomprises: at least one lit portion of the bioreactor system configuredto contain a culture of mixotrophic microorganisms in an aqueous culturemedium in an inner volume and expose the culture of mixotrophicmicroorganisms in the inner volume to at least some light from a lightsource; at least one dark portion of the bioreactor system in fluidcommunication with the at least one lit portion, the at least one darkportion configured to contain the culture of mixotrophic microorganismsin an aqueous culture medium in an inner volume in the absence of light;at least one organic carbon supply device configured to supply organiccarbon to a culture of mixotrophic microorganisms; and a circulationsystem configured to circulate the culture of mixotrophic microorganismsbetween the at least one lit portion and the at least one dark portion.

In some embodiments, the at least one lit portion may comprise at leastone selected from the group consisting of: a tank, a trough, a pond, anda raceway pond. In some embodiments, the at least one dark portion maycomprise at least one selected from the group consisting of: a foamfractionation device, a centrifuge, an electrodewatering device, a gasexchange device, and a contamination device. In some embodiments, the atleast one lit portion of the bioreactor system comprises a layer of theinner volume of a bioreactor which light penetrates and the at least onedark portion of the bioreactor system comprises a layer of the innervolume of the same bioreactor in which light does not penetrate.

In some embodiments, the bioreactor system may be an open system. Insome embodiments, the bioreactor system is a closed system. In someembodiments, the circulation system comprises at least one selected fromthe group consisting of a pump, a submersible thruster, and apaddlewheel. In some embodiments, the bioreactor system furthercomprises at least one inorganic carbon supply device, at least one gassupply device, a cover over at least par to the bioreactor system, andcombinations thereof. The light source may comprise at least oneselected from the group consisting of natural light and an artificiallighting device.

In another embodiment of the invention, a mixotrophic bioreactor systemcomprises: an open raceway pond comprising an inner volume of aconsistent depth, the raceway pond comprising: two straight awayportions separated by a center wall and bounded by straight outer wallsand a floor, two U-bend portions connecting the two straight awayportions to form a continuous loop, and bounded by a curved outer walland a floor; at least one arched turning vane disposed within eachU-bend portion; at least one submersible thruster disposed in the innervolume between the center wall and the outer wall of at least one of thestraight away portions and suspended from above by a support structurein the inner volume a distance from the floor; and at least one organiccarbon delivery device.

In some embodiments, the depth of the open raceway pond comprises 0.5 to10 meters. In some embodiments, the open raceway pond comprises a framestructure with a liner forming a surface of the floor, center wall, andouter walls of the open raceway pond. In some embodiments, the openraceway pond comprises a molded structure with a polymer forming asurface of the floor, center wall, and outer walls of the open racewaypond. In some embodiments, the at least one submersible thruster isdisposed at an end of the straight away portion within 20% of the lengthof the straight away portion. In some embodiments, the at least onesubmersible thruster is suspended a distance from the floor of 10-50% ofa height of an aqueous culture volume disposed in the inner volume ofthe open raceway pond.

In some embodiments, the open raceway pond further comprises at leastone heat exchanger. In some embodiments, the at least one heat exchangeris disposed in the at least one arched turning vane. In someembodiments, the at least one heat exchanger is disposed in the outerwalls, in the center wall, in the floor, or under the floor of the openraceway pond. In some embodiments, the at least one organic carbondelivery device comprises a pH auxostat system.

In some embodiments, the open raceway pond further comprises at leastone dissolved oxygen delivery device selected from the group consistingof a sparger tube, a membrane lining at least part of the floor of theraceway pond, a microbubble generator, an oxygen concentrator, a liquidoxygen injector, an oxygen saturation cone, and venturi injection by afoam fractionation device. In some embodiments, the open raceway pondfurther comprises a cover over at least part of the open raceway pond,at least one light source selected from the group consisting of naturallight and an artificial lighting device, and combinations thereof.

In another embodiment of the invention, a turning vane comprises: arigid structure comprising a height, width, and curvature forming anarched planar surface; and at least one functional component combinedwith the arched rigid structure. In some embodiments, the at least onefunctional component comprises an interior cavity configured to receivedand circulate a heat exchanger fluid. In some embodiments, the at leastone functional component comprises means for delivering at least oneselected from the group of organic carbon, nutrients, and gases. In someembodiments, the at least one functional component comprises anartificial lighting device. In some embodiments, the at least onefunctional component comprises at least one sensor. In some embodiments,the at least one functional component comprises a combination of two ormore selected from the group consisting of a heat exchanger, an organiccarbon delivery device, a nutrient delivery device, a gas deliverydevice, an artificial lighting device, and a sensor.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a perspective view of an open raceway pond bioreactorembodiment with arched turning vanes and submerged thrusters.

FIG. 2 shows a top view of an open raceway pond bioreactor embodimentwith arched turning vanes and submerged thrusters.

FIG. 3 shows a side view of an open raceway pond bioreactor embodimentand identifies the location of cross section A.

FIG. 4 shows a front view of an open raceway pond bioreactor embodimentat cross section A.

FIG. 5 shows a pair of arched turning vanes.

FIG. 6 shows a support structure for an arched turning vane.

FIG. 7 shows a perspective view of an open raceway pond bioreactorembodiment with multiple arched turning vanes and submerged thrusters.

FIG. 8 shows an embodiment of a multi-functional turning vane.

DETAILED DESCRIPTION Definitions

The term “microorganism” refers to microscopic organisms such asmicroalgae and cyanobacteria. Microalgae include microscopicmulti-cellular plants (e.g. duckweed), photosynthetic microorganisms,heterotrophic microorganisms, diatoms, dinoflagellattes, and unicellularalgae.

The terms “microbiological culture”, “microbial culture”, or“microorganism culture” refer to a method or system for multiplyingmicroorganisms through reproduction in a predetermined culture medium,including under controlled laboratory conditions. Microbiologicalcultures, microbial cultures, and microorganism cultures are used tomultiply the organism, to determine the type of organism, or theabundance of the organism in the sample being tested. In liquid culturemedium, the term microbiological, microbial, or microorganism culturegenerally refers to the entire liquid medium and the microorganisms inthe liquid medium regardless of the vessel in which the culture resides.A liquid medium is often referred to as “media”, “culture medium”, or“culture media”. The act of culturing is generally referred to as“culturing microorganisms” when emphasis is on plural microorganisms.The act of culturing is generally referred to as “culturing amicroorganism” when importance is placed on a species or genus ofmicroorganism. Microorganism culture is used synonymously with cultureof microorganisms.

Microorganisms that may grow in mixotrophic culture conditions includemicroalgae, diatoms, and cyanobacteria. Non-limiting examples ofmixotrophic microorganisms may comprise organisms of the genera:Agmenellum, Amphora, Anabaena, Anacystis, Apistonema, Pleurochyrsis,Arthrospira (Spirulina), Botryococcus, Brachiomonas, Chlamydomonas,Chlorella, Chlorococcum, Cruciplacolithus, Cylindrotheca, Coenochloris,Cyanophora, Cyclotella, Dunaliella, Emiliania, Euglena, Extubocellulus,Fragilaria, Galdieria, Goniotrichium, Haematococcus, Halochlorella,Isochyrsis, Leptocylindrus, Micractinium, Melosira, Monodus, Nostoc,Nannochloris, Nannochloropsis, Navicula, Neospongiococcum, Nitzschia,Odontella, Ochromonas, Ochrosphaera, Pavlova, Picochlorum,Phaeodactylum, Pleurochyrsis, Porphyridium, Poteriochromonas,Prymnesium, Rhodomonas, Scenedesmus, Skeletonema, Spumella, Stauroneis,Stichococcus, Auxenochlorella, Cheatoceros, Neochloris, Ocromonas,Porphiridium, Synechococcus, Synechocystis, Tetraselmis,Thraustochytrids, Thalassiosira, and species thereof.

The organic carbon sources suitable for growing a microorganismmixotrophically or heterotrophically may comprise: acetate, acetic acid,ammonium linoleate, arabinose, arginine, aspartic acid, butyric acid,cellulose, citric acid, ethanol, fructose, fatty acids, galactose,glucose, glycerol, glycine, lactic acid, lactose, maleic acid, maltose,mannose, methanol, molasses, peptone, plant based hydrolyzate, proline,propionic acid, ribose, sacchrose, partial or complete hydrolysates ofstarch, sucrose, tartaric, TCA-cycle organic acids, thin stillage, urea,industrial waste solutions, yeast extract, and combinations thereof. Theorganic carbon source may comprise any single source, combination ofsources, and dilutions of single sources or combinations of sources.

The terms “mixotrophic” and “mixotrophy” refer to culture conditions inwhich light, organic carbon, and inorganic carbon (e.g., carbon dioxide,carbonate, bi-carbonate) may be applied to a culture of microorganisms.Microorganisms capable of growing in mixotrophic conditions have themetabolic profile of both phototrophic and heterotrophic microorganisms,and may use both light and organic carbon as energy sources, as well asboth inorganic carbon and organic carbon as carbon sources. Amixotrophic microorganism may be using light, inorganic carbon, andorganic carbon through the phototrophic and heterotrophic metabolismssimultaneously or may switch between the utilization of each metabolism.A microorganism in mixotrophic culture conditions may be a net oxygen orcarbon dioxide producer depending on the energy source and carbon sourceutilized by the microorganism. Microorganisms capable of mixotrophicgrowth comprise microorganisms with the natural metabolism and abilityto grow in mixotrophic conditions, as well as microorganisms whichobtain the metabolism and ability through modification of cells by wayof methods such as mutagenesis or genetic engineering.

The terms “phototrophic”, “phototrophy”, “photoautotrophy”,“photoautotrophic”, and “autotroph” refer to culture conditions in whichlight and inorganic carbon (e.g., carbon dioxide, carbonate,bi-carbonate) may be applied to a culture of microorganisms.Microorganisms capable of growing in phototrophic conditions may uselight as an energy source and inorganic carbon (e.g., carbon dioxide) asa carbon source. A microorganism in phototrophic conditions may produceoxygen.

The terms “heterotrophic” and “heterotrophy” refer to culture conditionsin which organic carbon may be applied to a culture of microorganisms inthe absence of light. Microorganisms capable of growing in heterotrophicconditions may use organic carbon as both an energy source and as acarbon source. A microorganism in heterotrophic conditions may producecarbon dioxide.

The term “axenic” describes a culture of an organism that is entirelyfree of all other “contaminating” organisms (i.e., organisms that aredetrimental to the health of the microalgae or cyanobacteria culture).Throughout the specification, axenic refers to a culture that wheninoculated in an agar plate with bacterial basal medium, does not formany colonies other than the microorganism of interest. Axenic describescultures not contaminated by or associated with any other livingorganisms such as but not limited to bacteria, cyanobacteria, microalgaeand/or fungi. Axenic is usually used in reference to pure cultures ofmicroorganisms that are completely free of the presence of otherdifferent organisms. An axenic culture of microalgae or cyanobacteria iscompletely free from other different organisms.

The term “pH auxostat” refers to the microbial cultivation techniquethat couples the addition of fresh medium (e.g., medium containingorganic carbon such as acetic acid) to pH control. As the pH drifts froma given set point, fresh medium is added to bring the pH back to the setpoint. The rate of pH change is often an excellent indication of growthand meets the requirements as a growth-dependent parameter. The feedwill keep the residual nutrient concentration in balance with thebuffering capacity of the medium. The pH set point may be changeddepending on the microorganisms present in the culture at the time. Themicroorganisms present may be driven by the location and season wherethe bioreactor is operated and how close the cultures are positioned toother contamination sources (e.g., other farms, agriculture, ocean,lake, river, waste water). The rate of medium addition is determined bythe buffering capacity and the feed concentration of the limitingnutrient and not directly by the set point (pH) as in a traditionalauxostat. The pH auxostat is robust but controls nutrient concentrationindirectly. The pH level represents the summation of the production ofdifferent ionic species and ion release during carbon and nutrientuptake. Therefore the pH level can move either up or down as a functionof growth of the microorganisms. The most common situation is pHdepression caused by organic acid production and ammonium uptake.However, for microorganisms growing on protein or amino acid-rich media,the pH level will rise with growth because of the release of excessammonia.

Overview

When culturing microorganisms in mixotrophic conditions, the applicationof light, inorganic carbon, and organic carbon provides multiple cultureinputs, including energy sources that may be utilized by both the targetmicroorganisms (e.g., microalgae and cyanobacteria) and contaminatingorganisms (e.g., fungi, bacteria, rotifers, ciliates). The presence ofcontaminating organisms, balance of dissolved gases, and availability ofnutrients and energy sources did not have to be addressed at thelaboratory scale where small volumes, short culture durations, andindoor controlled conditions were utilized.

In an outdoor large scale mixotrophic production system, the potentialfor contaminating organisms to inhibit the target microorganisms in theculture may influence two pathways for large scale production. The firstpathway comprises an open or semi-closed system which does not operatein axenic conditions. In such non-axenic systems, the production systemand culturing methods may be designed to handle the volume and diversityof contaminating microorganisms. The second pathway comprises a closedsystem which operates in axenic conditions. In such axenic systems, theproduction system may be designed to maintain the proper cultureconditions in a closed system with the absence of contaminatingorganisms. In some embodiments, the bioreactors described may beoperated in axenic conditions. In some embodiments, the bioreactorsdescribed may operate in non-axenic conditions. With the preference forsome microorganism species to grow only in non-axenic conditions, theopen and non-axenic embodiments described herein provide the potentialfor culturing a broader scope of microorganisms in mixotrophicconditions than is available for systems using axenic fermenters.

A mixotrophy bioreactor system may comprise a culturing vessel with aninner volume configured to contain an aqueous culture of mixotrophicmicroorganisms, at least one lighting device or a component (e.g., anopening or window with some degree of transparency) to allow the innervolume exposure to at least some light (natural, artificial, or acombination thereof), and an organic carbon supply device. In someembodiments, the mixotrophy bioreactor system may further comprise asupply of inorganic carbon (e.g., carbon dioxide, bi-carbonate). In someembodiments, the mixotrophy bioreactor systems may further comprise: anautomated sensor and controls system; a programmable logic controlsystem; at least one sensor for detecting culture parameters such astemperature, pH, dissolved oxygen, dissolved carbon dioxide, flow rate,turbidity, and photopigments or carotenoids; at least one component formixing and circulating the culture; a gas supply (e.g., air, oxygen,nitrogen); and at least one heat exchanger. In some embodiments, themixotrophy bioreactor system may be disposed at least partiallyoutdoors. In some embodiments, the mixotrophy bioreactor system may bedisposed at least partially indoors. In some embodiments, the mixotrophybioreactor system may comprise a cover at least partially shielding themicroorganism culture from environmental elements such as light,temperature, heat, wind, air borne particles, and precipitation. In someembodiments, the bioreactor may comprise a culturing vessel such as, butnot limited to, a tank, bag, pond, raceway pond, or trough configured toallow at least some exposure to an inner volume of the culturing vesselto artificial or natural light, and an organic carbon supply device.

Dissolved Oxygen (DO) Distribution

One challenge not addressed with the laboratory scale bioreactors is thedistribution of dissolved oxygen within the bioreactor volume. While amixotrophic microorganism may produce oxygen when utilizing light as anenergy source and inorganic carbon as a carbon source, oxygen isconsumed when utilizing organic carbon as an energy and carbon source.Therefore, maintaining a dissolved oxygen level in the culture isimportant for maintaining growth rates driven by the utilization oforganic carbon. In a small volume bioreactor, the dissolved oxygencontent may be relatively uniform across the depth of a mixotrophicculture. Additionally, a small volume bioreactor may be easilyilluminated in a substantially uniform manner, therefore allowing themetabolism of all microorganisms in the laboratory or bench top scalebioreactor to be functioning essentially in the same manner.

When the depth of the bioreactor is increased, a gradient distributionof dissolved oxygen may form over the depth of the bioreactor whenmixing does not sufficiently distribute the dissolved gases in a uniformmanner. The lighting in a large volume bioreactor also may not beuniform due to the depth of the bioreactor. In one non-limiting example,light may be available within a short distance (e.g., less than 10 cm)of the air/liquid interface of an open pond bioreactor and rely onmixing to periodically circulate the microorganism through the differentdepths of the culture volume to expose the microorganisms tointermittent light.

Below the light path distance, the available energy source would beorganic carbon in a mixotrophic culture. Therefore factors that maycontribute to the distribution of dissolved gases in a mixotrophicculture may comprise: 1) the mixing regime used in the bioreactorsystem, and the ability of the mixing regime to uniformly mix anddistribute dissolved gases; 2) the loss of gases at the air/liquidinterface (e.g., bursting of bubbles that do not dissolve in the aqueousmedium); 3) the location of the gas supply within the bioreactor system(e.g., at the deepest portion of the bioreactor, between the air/liquidinterface and the deepest portion of the bioreactor, within a turbulentflow region, within a laminar flow region); 4) the residence time of thesupplied gas bubbles within the culture; 5) the consumption rate ofgases by the microorganisms due to metabolic activity and availableenergy sources; and 6) the production of gases by the microorganisms dueto metabolic activity and available energy source.

The large scale bioreactor may circulate the mixotrophic microorganismsthrough the volume of the bioreactor and the distribution of dissolvedoxygen concentrations. The circulation through the depth of the largescale bioreactor may allow the mixotrophic microorganism to maximize theutilization of the available energy sources in the different locationsfor growth and product development without experiencing stress fromquickly changing culture conditions.

In some embodiments, the dissolved oxygen concentration in the culturemedium at the deepest portion of the bioreactor and at the air/liquidinterface may vary between 10 and 500%, including a concentration at thedeepest portion of 1 to 500% greater than the dissolved oxygenconcentration at the air/liquid interface. The difference in dissolvedoxygen concentration at the deepest portion and the air/interface may bethe largest when the air or oxygen introduction device is disposed at ornear the bottom of the reactor. In one non-limiting example, the depthof the bioreactor may be such that the dissolved oxygen concentrationmay vary between about 1 g/L and 1.1-5 g/L. In some embodiments, thedepth of the bioreactor may be greater than 0.5 meters. In someembodiments the depth of the aqueous culture in the bioreactor may bebetween 0.5 and 10 meters, and preferably between 0.5 and 2 meters. Inother embodiments, the mixing within the culture volume is sufficient todistribute the dissolved oxygen in a substantially uniform concentrationacross the depth of the culture volume, (e.g., within 10%).

Temperature Fluctuation

Another challenge not addressed by laboratory or bench top scalebioreactors in mixotrophic culture conditions with a small volume is thechange in temperature of the culture volume. Laboratory and bench topscale bioreactors may function with a small volume (i.e., thermal mass)because they are in a controlled environment for typically a shortduration of time. In a small volume bioreactor or a bioreactor with ashallow depth for light path purposes, the temperature of the culturemedium may be susceptible to large changes when placed in a commercialenvironment, such as outdoors where the night/day cycle, weather, andclouds may change the surrounding temperature substantially over aperiod of hours. Changes in temperature may stress the mixotrophicmicroorganisms, or decrease the efficiency of growth or productformation during periods when the temperature is outside of an optimalrange. When the temperature of the culture of microorganisms cannot beconsistently maintained on its own, methods of cooling and heating mayneed to be added to the bioreactor system resulting in lower energyefficiency and high capital costs for the system.

When the depth of the bioreactor is increased to hold a larger culturevolume, the larger thermal mass may be less sensitive to the temperatureswings caused by heating during the daylight and cooling during thenight. By design, the large scale mixotrophic bioreactor will have aculture volume with more thermal inertia than the laboratory and benchtop bioreactor designs, and thus may be subject to thermal gradients,both spatially and temporally. Some embodiments of these large scalebioreactors may by physically located outdoors thus being exposed tolarge variations in environmental conditions. In some embodiments, thelarge scale bioreactor may use methods of cooling and heating to aid inthe establishment of controlled growth environments and may use acontrol system to aid in thermal control.

In some embodiments, the bioreactor may comprise a sufficient volume anddepth to reduce the average temperature change over a 24 hour period toless than a 1-20° C. difference without the use of heat exchangers, andpreferably less than a 1-10° C. difference. In some embodiments, thedepth of the aqueous culture in the bioreactor may be greater than 0.5meters. In some embodiments the depth of the aqueous culture in thebioreactor may be between 0.5 and 10 meters, preferably between 0.5 and2 meters. Therefore, a large volume culture may not need aproportionally larger use of heat exchangers as a small volume cultureto maintain an optimal temperature due to the larger thermal mass of thelarge volume culture, and the amount of energy per volume of cultureneeded to control temporal changes in culture temperature may be lessthan small volume cultures.

In some embodiments, passive cooling, natural evaporative cooling, orevaporative cooling assisted by fan forced airflow over the surface ofthe microorganism culture volume may increase the thermal stability ofthe microorganism culture. Parameters that may affect a passive orevaporative cooling system on a bioreactor system may comprise: time ofday, day of year, geographic location, position of the sun, solarheating load, humidity, moisture content, temperature, water partialpressure, Fresenl-law reflectivity of a cover, transmissivity of acover, reflection within the bioreactor system, nocturnal re-radiationof energy between the bioreactor and sky, water evaporation, andbioreactor air flow turbulence. Heat exchangers in contact withbioreactor surfaces, culture volume, or combinations thereof may also beused to assist in the maintenance of a consistent temperature or effectspatial temperature distribution through heating or cooling.

Mixotrophic Bioreactor with Light and Dark Portions

Conventional bioreactors designed for phototrophic growth ofmicroorganisms in an aqueous culture focus on systems with short lightpaths. In an aqueous microorganism culture, light may only penetrate adistance as little as 2-5 cm into the culture. By using bioreactors withshort light paths, a larger percentage of microorganisms in thebioreactor system are exposed to light for energy in the photosynthesisprocess, and self-shading of the microorganisms may be mitigated. Shortlight paths may be achieved by reducing the depth of the culture ordistance that light has to penetrate, essentially dictating long andshallow or narrow reactors that must cover a large amount of surfacearea to provide high volumes.

Conventional bioreactors designed for heterotrophic growth ofmicroorganisms are not concerned with the availability of light and maycomprise larger and deeper volumes in a smaller footprint than acomparable volume of a phototrophic reactor. In these heterotrophicsystems, mechanical mixing and closing the system are important toensure that the microorganisms are: maintained in suspension; receivingthe administered dissolved oxygen and organic carbon; and preventing theintroduction of competing and contaminating organisms. While mechanicalmixing may be effective for distributing the organic carbon source andgas transfer, some types of mechanical mixing (e.g., open propellers,stirrers) may also impart a shear stress on the microorganisms that maypotentially harm the microorganisms if the shear stress level is abovethe tolerance level of the microorganisms. The flexibility ofmixotrophic microorganisms to utilize multiple energy sources allows amixotrophic bioreactor system to be less constrained in design by thelimiting features of light path, mixing devices, or closing of thesystem.

In some embodiments a mixotrophic bioreactor system for culturingmicroorganisms in an aqueous culture medium may comprise an organiccarbon supply device, at least one lit portion receiving at least somelight, and at least one dark portion receiving no light. In someembodiments, the at least one lit portion and the at least one darkportion may be components of a single apparatus, such as a deepbioreactor in which the lit portion may be layered on top of or belowthe dark portion. In some embodiments, the at least one lit portion andthe at least one dark portion may be separate apparatuses connected influid communication. In some embodiments, the bioreactor system maycomprise at least one portion that is open. In some embodiments, thebioreactor system may comprise at least one portion that is closed. Theculture of mixotrophic microorganisms may be circulated between the atleast one lit portion and at least one dark portion by any known meanssuch as, thrusters, pumps, paddlewheels, and gravity. In someembodiments, the lit and dark portions may be the physical components ofthe bioreactor system, with the lighted and dark nature controlled bythe timing and use of artificial light devices.

In an alternate embodiment, the method of mixing may enable a verticalflow pattern such that the fluid may be swept from top to bottom in arotating fashion to bring the cells to the light at a faster frequency.In one non-limiting example, the use of a submerged thruster creates arotation, vertical, or swirling flow that brings the microalgae orcyanobacteria cells to the air/liquid interface of the aqueous cultureto provide exposure to light (i.e., lit portion) several times beforereturning to a depth of the aqueous culture comprising a dark portion.One advantage of providing additional light in a mixotrophic culture ofmicroalgae or cyanobacteria is the reduction of carbon energy to drivegrowth, formation of pigments, formation of proteins, formation otherproducts that are preferentially formed in the presence of light, andcombinations thereof.

In some embodiments, the circulation between the at least one litportion and the at least one dark portion create a light duty cycle formixotrophic microorganisms of 2 to 25%, and preferably 5%. The lightduty cycle is defined as the fraction of a total light-dark microcyclein which an individual microorganism is exposed to light. The light dutycycle is calculated by dividing the time the microorganisms are exposedto light by the total time the microorganisms spend in the bioreactorsystem. The calculated light duty cycle is expressed as a percentage. Insome embodiments, the bioreactor may comprise a plurality ofstrategically spaced lit and dark portions to create a repeating lightduty cycle when the culture of microorganisms is circulated, which maybe controlled by the flow rate of the culture of microorganisms. In someembodiments, the light duty cycle may also be controlled by the timingand use of artificial lighting devices.

The at least one lit portion exposes the culture of mixotrophicmicroorganisms to at least some light from a light source. In someembodiments, the light source may comprise at least one lighting deviceproviding artificial light. The at least one artificial lighting devicemay comprise any lighting device capable of providing light to a cultureof microorganisms such as, but not limited to, fluorescent tubes, lightemitting diodes (LED), micro LEDs, high pressure sodium lamps, highintensity discharge lamps, neon lamps, metal vapor lamps, halogen lamps,sulfur plasma lamps, and incandescent bulbs. In some embodiments, the atleast one lighting device may be selected or tuned to provide light of aparticular wavelength spectrum or combination of spectrums such as, butnot limited to, violet (about 380-450 nm), blue (about 450-495 nm),green (about 495-570 nm), yellow (about 570-590 nm), orange (about590-620 nm), red (about 620-750 nm), and far red (about 700-800 nm),infrared (IR) (about 1,000-20,000 nm) and ultraviolet (UV) (about 10-400nm). In some embodiments, the application of light may be continuous,discontinuous, flashing, or pulsing to create any desired light/darkcycle. In some embodiments, the intensity of light supplied by the atleast one lighting device may comprise a constant intensity or variableintensity. The at least one lighting device may be mounted anywhere onthe bioreactor module, suspended or submerged in the culture volume, ormay be separate from the bioreactor module.

In some embodiments, the light source may comprise natural light such assunlight. In some embodiments, the lit portion receiving sunlight maycomprise a cover configured to block at a least a portion of thesunlight from contacting the culture of microorganisms. In someembodiments, the cover may block between 5-95% of light. The cover maycomprise a semi-transparent photovoltaic panel, a film which selectivelyblocks light in a specific wavelength range, a passive shade cloth(e.g., an aluminet shade cloth, a shade cloth that blocks some light orblocks specific wavelengths), a semitransparent polymer, tinted glass,and combinations thereof. In some embodiments, the lit portion may be aportion of a large volume and deep pond, trough, or tank in which lightpenetrates such as, but not limited to, the air/water surface interfaceand top fraction of a deep pond or tank experiencing light penetration.The at least one lit portion may receive light at 100-2,500 μmolphotons/m² s, preferably 200-500 μmol photons/m² s. In some embodiments,the light source may comprise a combination of at least one lightingdevice providing artificial light and natural light (e.g., sunlight).

The at least one dark portion comprises the absence of light for theculture of mixotrophic microorganisms. In some embodiments, the at leastone dark portion may comprise a cover of opaque material configured toprevent light from contacting the culture of microorganisms. In someembodiments, the at least one dark portion may comprise the depth of alarge volume below where light may penetrate. In some embodiments, theat least one dark portion may comprise a functional apparatus in fluidcommunication with the at least one lit portion. The functionalapparatus may comprise a foam fractionation device (e.g., proteinskimmer, bubble column, dissolved air flotation tank), centrifuge,electrodewatering device (e.g., reactor exposing the culture to anelectric field), dewatering device (e.g., filtration apparatus,sedimentation tank), contamination control device (e.g., device forapplying ozone or other contamination control solutions), gas exchangedevice (e.g., degassing tank), holding tank, and combinations thereof.

In one non-limiting example, the at least one dark portion comprises aprotein skimmer with adjustable settings which provides a plurality offunctions while shielding the culture of microorganisms from light, suchas: gas injection; de-gassing; and removal of foam and constituents suchas contaminating microorganisms, suspended solids, debris, and clumpedmicroorganisms above a threshold size from the culture through foamfractionation. The removal of foam and constituents from the culture mayreduce competition for resources with the mixotrophic microorganisms andextend the life of the culture of mixotrophic microorganisms.

Bioreactor System Embodiments

The following bioreactor system embodiments incorporate the describeddifferences between conventional phototrophic or heterotrophic systemsand mixotrophic systems, and small volume and large volume mixotrophiccultures regarding dissolved oxygen distribution, temperaturefluctuation, and access to light for successful mixotrophic culturing ata large scale in a bioreactor system with lit and dark portions. In afirst non-limiting embodiment, a large scale mixotrophic bioreactorsystem configured for culturing microorganisms in an aqueous culturemedium may comprise a raceway pond, trough, or tank bioreactor providinga lit bioreactor configured for containing an aqueous culture in aninner volume that receives at least some light from a light source, anda foam fractionation apparatus (e.g., protein skimmer, bubble column)providing the dark portion of the bioreactor system with an opaque tanksection configured for containing an aqueous culture in an inner volumethat receives no light. The lit bioreactor may be in fluid communicationwith the foam fractionation apparatus through conduits attached to aninlet and outlet of the lit bioreactor to circulate the aqueous culturemedium between lit and dark portions. The depth of the lit bioreactormay be designed for a culture volume size with a sufficient thermal massto aid in the control of temperature fluctuations

The culture of microorganisms may be circulated by pumps, paddlewheels,or thrusters in the lit bioreactor, and upon exiting the lit bioreactorthrough an outlet the culture flows to the foam fractionation apparatus.In some embodiments, gas injection (e.g., oxygen, air, carbon dioxide,nitrogen) for dissolved gas manipulation may be performed by the foamfractionation apparatus by venturi injection, sparger, air muffler,microbubble generator, and the like. Upon exiting the foam fractionationapparatus, the culture returns to the lit bioreactor. Using the gasinjection of the foam fractionation apparatus may aid in controlling thedissolved oxygen concentration by allowing the gas to be injected at asingle point where the residence time can be controlled by the flow ratethrough the circulation back to the lit bioreactor where the aqueousmedia may be thoroughly mixed. In some embodiments, organic carbon maybe dosed by a metering pump into the discharge line of the foamfractionation apparatus, which returns the culture to the lit bioreactorand completes the culture circulation path within the bioreactor system.In some embodiments, the organic carbon may be dosed directly into thelit bioreactor, or dosed directly within the foam fractionationapparatus. In some embodiments, the organic carbon may comprise aceticacid and may be dosed using a pH auxostat system.

In some embodiments, the culture parameters (e.g., pH, temperature,dissolved oxygen, dissolved carbon dioxide) may be detected by probesand sensors at various locations along the circulation path such as, butnot limited to: within the foam fractionation device, at the inlet ofthe foam fractionation device, at the outlet of the foam fractiondevice, and within the lit bioreactor. In some embodiments, the litbioreactor may be at least partially covered with a cover that blocks atleast some light. In some embodiments, the cover may comprise a canopyor a greenhouse. In some embodiments, the cover may comprise a lowprofile cover. In some embodiments, the cover may comprise a materialthat blocks transmission of between 1% and 99% of light to the culture,such as but not limited to a passive shade cloth. In some embodiments,the cover may comprise a film which selectively blocks transmission ofcertain wavelengths of light to the culture, or semitransparentphotovoltaic panels.

In some embodiments, at least one fan may be disposed in the cover ofthe system to facilitate forced air circulation across the surface ofaqueous culture and the head space between the cover and the surface ofthe aqueous culture. In some embodiments, the circulation of the aqueousculture through the bioreactor system may be adjusted to a desired dutycycle comprising the amount of time the culture spends in the foamfractionation apparatus compared to the total time in the bioreactorsystem.

In some embodiments, gases may be supplied to the culture throughaeration tubing disposed within the lit bioreactor in addition to thegasses supplied by the foam fractionation apparatus. In someembodiments, a heat exchanger (e.g., coils fed with heat exchangerfluid) may be submerged in the culture volume of the lit bioreactor orin the opaque tank section of the foam fractionation apparatus. The foamfractionation apparatus may also provide the function of removing foamfrom the aqueous culture, as foam has been known to harbor contaminationand thus removal of the foam helps to maintain the health of themicroorganism culture.

In a further embodiment of the lit bioreactor, the bioreactor maycomprise an open raceway pond with two straight away portions separatedby a center wall, two U-bend portions connecting the straight awayportions into a closed loop, at least one organic carbon deliverydevice, and at least one submersible thruster to provide the mixing andcirculation of the aqueous culture through the closed loop. The straightaway portions and U-bend portions of the open raceway pond form acontinuous looped fluid circulation path for the aqueous culture definedby outer wall surfaces of the U-bend and straight away portions and theouter surfaces of the center wall. Along this continuous looped fluidcirculation path, the culture is provided with at least some light,nutrients, and organic carbon. The open raceway pond may receive lightfrom a natural light source (e.g., sunlight), an artificial lightsource, or combinations thereof. The open raceway pond may beconstructed above ground with a frame or molded body, or may beconstructed in the ground.

In some embodiments, the width of the open raceway pond may compriseabout 3 to 12 meters (about 10 to 40 feet) total and preferably about 9meters (about 30 feet), with the length dependent on the desired culturevolume. In some embodiments, the height of the bioreactor may be 1 to 12meters to allow for a culture depth of 0.1 to 10 meters, preferablybetween 0.5 and 2 meters. In some embodiments, the culture may bestarted at a first depth and then increase to a maximum culture depth asthe culture density increases after inoculation. The depth of the openraceway pond bioreactor incorporates the concepts previously describedof a dark portion layered with lit portions in the same culture volume(e.g., a dark portion below the distance light penetrates the topculture surface, a dark portion beyond the distance light from submergedlighting devices reaches within the culture volume), and the thermalstability of a larger culture volume to reduce or eliminate therequirements for heat exchangers.

In some embodiments, the center wall separating the straight awayportions may be about 0.1-0.6 meters (about 6-24 inches) in width,preferably about 0.25 meters (about 10 inches), and of a height whichprotrudes above the depth of the aqueous culture. The floor of the openraceway pond may be flat, contoured, or combinations thereof. In someembodiments, the contoured floor of the raceway pond is V or U shaped.In some embodiments, the floor may be flat to create an inner volume ofthe open raceway pond with a consistent depth along the looped cultureflow path.

A series of different sized open raceway pond bioreactors may be usedtogether in a system for culturing mixotrophic microalgae, with thevolume of the ponds increasing to accommodate the increase of culturedensity at different stages. A first pond bioreactor may comprise aculture volume of 15,000 to 20,000 liters. A second pond bioreactor maycomprise a culture volume of 100,000 to 130,000 liters. A third pondbioreactor may comprise a culturing volume of over 500,000 liters. Thedepth may be the same for each pond bioreactor, regardless of thevolume, but the width and length may change for the different volumes.For example, a 100,000 liter pond bioreactor may comprise a width ofabout 4.5 meters (about 15 feet) and a length of about 27 meters (about89 feet) comprising a width to length ratio of about 1:6; and a 500,000liter pond bioreactor may comprise a width of about 9 meters (about 30feet) and a length greater than 27 meters (about 90 feet).

In some embodiments, the open raceway pond may be molded from a polymeras one piece or in sections that are coupled together. In someembodiments, the raceway pond may comprise a rigid frame covered with aliner material. The liner material may be selected to resist degradationcaused by low pH culture solutions, the organic carbon, and otherconstituents of the microorganism culture. Examples of suitable linersinclude Lake Tahoe liner, F-Clean NEW soft-shine white (100 μm), Raven(821 W Algonquin St, Sioux Falls, S.Dak. 57104) 20 mil grey/black, Raven20 mil white/white, and Western Environmental Liner (8121 W. Harrison,Tolleson, Ariz. 85353) polypropylene liner (45 mil). The rigid frame maycomprise wood, plastic, metal, and similar suitable materials.

In some embodiments, the bioreactor system may comprise at least onearched turning vane in each of the U-bend portions, and may comprise twoor more turning vanes in each of the U-bend portions dependent on thevolume and size of the bioreactor. The turning vanes comprise a height,width, and curvature forming an arched planar surface for guiding theflow of the aqueous microorganism culture. The turning vanes aredesigned to facilitate the flow of the aqueous culture through theU-bend portions, and change the direction of the flow to follow the 180degree turns into the straight away portion upon exiting the U-bendportion. In some embodiments, the downstream end of the turning vane maycomprise an asymmetrical curved design extending past the beginningposition of the upstream portion of the turning vane and into thebeginning of the straight away portion. In some embodiments, the turningvanes comprise a symmetrical curved design. In some embodiments, theupstream end of the turning vane may begin wherein the straight awayportion ends and the U-bend portion begins. In some embodiments, theturning vanes may also create a passive vortex through the end-wallboundary layer migration that aids in mixing the aqueous culture duringcirculation. In some embodiments, the turning vanes disposed in the sameor different U-bend portions may have the same curvature profile. Insome embodiments, the turning vanes disposed in the same or differentU-bend portions may have different curvature profiles, including groupsof the turning vanes in the same U-bend portion with different curvatureprofiles.

The height of the turning vanes may be greater than the depth of theaqueous culture. In some embodiments, the turning vanes may be securedto the floor of the open raceway pond and at the top to the center walland outer walls of the U-bend portion by support members, and secured atthe base to the floor of the U-bend portion. The turning vanes andsupport members may comprise suitable polymers or metals (e.g.,stainless steel) suitable for microorganism culturing formed by smoothsolid material, or rigid frames with a surface comprising linermaterial, polymer sheets, or sheets of metal mounted to the frames.

One design emphasis for the open raceway pond bioreactor is theminimization of the equipment disposed within the culture volume thatmay provide a surface for contamination to accumulate and proliferate.With the turning vanes already disposed in the culture volume, at leastone other functional component may be added to, combined with, orintegrated with the rigid structure of the turning vanes to providefunctionality beyond guidance of fluid flow and minimize the number ofseparate components disposed in the culture volume. In some embodiments,the rigid structure of the turning vanes may comprise an arched rigidstructure frame with a material forming the surface such as, but notlimited to, a liner material, polymer, or sheet metal. The materialcovering the frame provides sufficient spacing within the frame to houseat least one functional component. The rigid frame may comprise wood,metal, plastic, or similar suitable materials. In some embodiments, theat least one functional component may form the surface of the turningvane.

In some embodiments, the at least one other functional component maycomprise a heat exchanger, such as but not limited to tubular or plateheat exchangers configured to receive and circulate heat exchangerfluid. In some embodiments, the at least one other functional componentmay comprise a device such as, but not limited to conduit, nozzles,injectors, bubblers, and pumps for delivering a nutrient medium, organiccarbon, or other nutrients. In some embodiments, the at least one otherfunctional component may comprise a device such as, but not limited toconduit, nozzles, injectors, bubblers, and pumps for delivering gases(e.g., oxygen, carbon dioxide, air). In some embodiments, the at leastone other functional component may comprise an artificial lightingdevice such as, but not limited to LEDs. In some embodiments, the atleast one other functional component may comprise sensors or probes.

In some embodiments, the turning vanes may comprise an arched rigidstructure that does not include a separate frame, such as a singlepiece, two piece, formed, or molded structure. In some embodiments, theat least one other functional component may be integrated with theframeless rigid structure of the turning vane. Integrating the at leastone other functional component into the rigid structure may reduce thethickness of the turning vane compared to a turning vane comprising aframe. In one non-limiting embodiment, the turning vane may comprise anarched structure comprising a cavity, such as sheets of material (e.g.,metal, plastic) joined at the edges to form an interior cavity that maycirculate or serve as a conduit for a fluid (e.g., heating fluid, gas,organic carbon solution, nutrient solution). The surfaces of the sheetsof material may be smooth or contoured.

In some embodiments, the integrated structure of the turning vane mayonly contain openings for allowing a fluid to be introduced into theinterior cavity from a reservoir, circulated with the cavity, andreturned to the reservoir, such as for heat exchanger fluid thatprovides a function by circulation within the cavity for heat transferwith a the culture without introduction into the culture volume. Thecavity may comprise additional internal partitions to guide exchangefluid through a flow path or distribute the fluid evenly throughout thecavity. In some embodiments, the integrated structure of the turningvane may comprise additional openings allowing the fluid to exit theinterior cavity for introduction into the culture volume, such as whenthe interior cavity serves as a conduit for introducing a gas, organiccarbon, or nutrients into the culture volume. In some embodiments, themulti-functional turning vane may comprise a combination of functionsbeyond the guidance of fluid flow such as a combination of two or morefunctions selected from the group consisting of a heat exchange, organiccarbon delivery, nutrient delivery, gas delivery, artificial lighting,and parameter sensing.

In some embodiments, the bioreactor system may comprise plate or tubularheat exchangers disposed in the raceway pond underneath the liner, inthe outer walls, in the center wall, in the floor, under the floor, andcombinations thereof. In some embodiments, the heat exchangers maycomprise a combination of heat exchangers disposed within the turningvanes and in the raceway pond underneath the liner, in the outer walls,in the center wall, in the floor, or under the floor. By positioning theheat exchanger within the turning vane or underneath the liner, thenumber of components submerged in the aqueous culture on whichcontamination may grow on or adhere to may be minimized, thus providinga healthier environment for the culture of microorganisms. Depending onthe location of the heat exchangers, a plate heat exchanger disposedwithin or under a raceway pond bioreactor surface may provide moresurface area for heat exchange than a tubular heat exchanger. A heatexchange fluid for circulation in the heat exchangers to cool or heatthe aqueous culture may be provided by conduits between the heatexchangers and a fluid reservoir. In other embodiments, the bioreactormay comprise a heat exchanger (e.g., a tube and shell heat exchanger)disposed outside of the bioreactor volume through which a volume of theaqueous culture is circulated through for heat exchange.

Submersible thrusters are commercially available from a number ofsources, such as Xylem (1 International Drive, Rye Brook, N.Y., 10573)which produces the Flygt brand of submersible jet ring thrust mixerproducts. The number of submersible thrusters may be dictated by thevolume of the aqueous culture and length of the straight away sectionsof the open raceway pond, and may comprise at least 1, at least 2, atleast 4, at least 8, or more to sufficiently mix the culture volume. Forexample an open raceway pond bioreactor with a 20,000 liter volume mayuse two submerged thrusters in total, consisting of a single thrusterdisposed at two different locations; and an open raceway pond bioreactorwith a 500,000 liter volume may use at least four submerged thrusters intotal, consisting of at least two thrusters disposed at least twodifferent locations.

In some embodiments, the submersible thrusters may be disposeddownstream of the turning vane in the U-bend portion of the racewayponds in positions such as, but not limited to, at the U-bend exits andmid channel in the straight away portions. In some embodiments, the atleast one submersible thruster may be disposed at an end of a straightaway portion near the U-bend portion within 20% of the length of thestraight away portion at the end. For example if the straight awayportion is 100 meters long, the at least one submersible thruster may bedisposed within the 20 meters of an end of the straight away portion(i.e., distance between the end of the straight away portion and thesubmersible thrusters is 20 meters or less).

In some embodiments, the at least one submersible thruster may bedisposed equidistant from the center wall and the outer wall of thestraight away portion. In some embodiments, the at least one submersiblethruster may be disposed in the straight away portion equidistancebetween the two U-bend portions. In some embodiments, multiplesubmersible thrusters at a single location in the bioreactor may be inparallel at the same axial position, offset from each other, orstaggered depending on the desired fluid movement. In some embodiments aplurality of submersible thrusters may be disposed on opposite side orends of the bioreactor, or at intervals along the length of thebioreactor

By positioning the submersible thrusters at the U-bend exit the thrustproduced may be maximized in the straight line flow of the culturethrough the straight away portions and momentum may be added to theculture flow exiting the U-bend portions where some velocity of theculture flow may be lost due change in flow direction. In someembodiments, the at least one submersible thruster may be suspended inthe inner volume of the open raceway pond a distance from the floor ofthe open raceway pond from above by a support structure. In someembodiments, the thrusters may be disposed at a distance measured fromthe floor of the raceway pond that is 10-50% of the culture volumeheight, preferably at a position measured from the floor of the racewaypond about 20-30% of the height of the aqueous culture volume. Forexample, if the culture volume height is 2 meters, the submersiblethruster may be positioned between 0.2-1 meters above the floor. Thedepth positioning of the thrusters in the raceway pond also facilitatesmixing the culture so that the microorganisms at the bottom of theculture pond depth are circulated to the air/liquid interfaceperiodically.

The support structure suspending the submersible thrusters from abovemay comprise support members coupled to the center wall and outer wallsof the bioreactor. In some embodiments, the support structure maycomprise multiple support members coupled together in a manner (e.g.,sliding and locking, discrete locking positions, friction fits,clamping) to allow the position of the submersible thruster to beadjusted vertically (i.e., in the depth of the culture volume) andhorizontally (i.e., between the outer and center walls). By utilizingthrusters that may be suspended from a support structure above theculture volume, the thruster provides an advantage over a submergedtraditional propeller mixer that is fixed in place with the motorlocated outside the raceway pond. A traditional propeller mixer isfastened to a rotating shaft which is driven by a motor, and in araceway pond the shaft needs to be parallel to the flow direction, whichwould require the shaft to be submerged by penetrating one of the wallsto connect to the motor disposed outside the pond. Penetration of thepond wall provides an opportunity for a leak and also limits theadjustment of the propeller positioning. A completely submersiblethruster that is suspended from above may be adjusted vertically andhorizontally more easily for optimal placement in culture volume formixing than a propeller fixed in position through a pond wall.Additionally, the suspended submersible thruster does not introduceadditional opportunities for a leak in the open raceway pond outer wall.

The submersible thrusters may be sized based on the culture volume sizeand size of the raceway pond bioreactor to sufficiently mix the cultureover the entire depth of the raceway pond bioreactor and propel theculture. For example, in a 20,000 liter pond bioreactor the thrustersmay be sized to produce 25 pounds of thrust. In cultures of shearsensitive microalgae or cyanobacteria, the submersible thruster may bereplaced with a different mixing device, such as a paddlewheel.

An organic carbon source may be dosed within the bioreactor at multiplepoints by at least one organic carbon delivery device. In someembodiments, the organic carbon may be dosed at a location upstream ofthe mixing device (e.g., thruster, paddlewheel, pump) to allow theorganic carbon to be sufficiently mixed in the aqueous culture.

In some embodiments, the dosed organic carbon may be acetic acid. Infurther embodiments, the dosing of acetic acid may be conducted with apH auxostat system to both control pH and maintain grow rates of themicroorganism. For example, the acetic acid flow rate may comprise 6.3L/min of 20% acetic acid in order to achieve a growth rate of 6 g/L-daywith a culture of Chlorella. In some embodiments, the dosed organiccarbon source may be glucose, glycerol, or any other suitable organiccarbon source depending on the microorganism. In some embodiments, theat least one organic carbon delivery device may comprise an outletdisposed within the open raceway pond configured to deliver organiccarbon to the aqueous culture. In some embodiments, the at least oneorganic carbon delivery device may comprise an outlet disposed above theopen raceway pond configured to deliver organic carbon to the aqueousculture. In some embodiments, the organic carbon may be deliveredthrough a multi-functional turning vane.

In some embodiments, gases such as air, oxygen, carbon dioxide, andnitrogen, may be supplied to the culture in the bioreactor systemthrough devices to ensure maintenance of the desired dissolved gas levelsuch as: sparger tubes located at the bottom of the bioreactor, spargertubes located at the base of the inner diameter and outer diameter ofthe raceway pond, a membrane (e.g., Prototype Tyvek) lining the bottomof at least part of the raceway pond, a microbubble generator, an oxygenconcentrator, liquid oxygen injection, an oxygen saturation cone, amulti-functional turning vane, and combinations thereof. In someembodiments, a foam fractionation device, such as a protein skimmer withventuri injection may be in fluid communication with the bioreactorpond, and process the aqueous culture during circulation and introduceair or oxygen into the culture medium through venturi injection. In someembodiments, the oxygen supply devices may be sized to maintain thedissolved oxygen content above 3 mg/L in the aqueous culture ofmicroorganisms.

In some embodiments, the open raceway pond bioreactor may be at leastpartially covered with a cover that blocks at least some light. In someembodiments, the cover may comprise a canopy or a greenhouse. In someembodiments, the cover may comprise a low profile cover. In someembodiments, the cover may comprise a material that blocks transmissionof between 1% and 99% of light to the culture, such as but not limitedto a passive shade cloth. In some embodiments, the cover may comprise afilm which selectively blocks transmission of certain wavelengths oflight to the culture. In some embodiments, the cover may comprisesemi-transparent photovoltaic panels.

In some embodiments, the bioreactor system may comprise probes orsensors to measure and monitor at least one of pH, temperature, NO₃,dissolved oxygen, dissolved carbon dioxide, turbidity, cultureconcentration, flow velocity, flow rate, light, and photopigments orcarotenoids. The probes or sensors maybe located at one or multiplelocations within the bioreactor system and disposed in mid-depth in theculture volume. In some embodiments, the pH sensors are used to controlpH by organic carbon addition at locations directly upstream of thesubmersible thrusters. NO₃ may be added as needed manually or withautomated equipment based on the values measured by the probes orsensors.

One non-limiting embodiment of the open raceway pond bioreactor is shownin FIGS. 1-4. The open raceway pond bioreactor 100 comprises an outerwall 101, center wall 102, arched turning vanes 103, submerged thrusters104, and support structure 105 (horizontal), 106 (vertical) or thesubmerged thrusters. The outer wall 101 and the center wall 102 form theboundaries of the straight away portions 120 and U-bend portions 130 ofthe bioreactor in FIGS. 1-4. In FIGS. 1-4 the center wall 102 is shownas a frame for viewing purposes, but in practice panels are insertedinto open sections of the frame or a liner placed over the frame to forma solid center wall surface. Also, the outer wall 101 of the bioreactoris FIGS. 1-4 is depicted as multiple straight segments connected atangles to form the curved portion of the U-bend 130, but the outer wall101 may also form a continuous curve or arc as shown in FIG. 7. FIG. 4shows a cut away view of the bioreactor 100 at cross section A asidentified in FIG. 3, which further displays the submerged thruster 104being disposed in the inner volume of the bioreactor a distance abovethe floor 115 of the bioreactor and spaced from the outer wall 101 andthe center wall 102.

FIG. 2 further shows the asymmetrical shape of the arched turning vanes103, first end 140 of the turning vane at the beginning of the U-bendportion 130 and the second end 141 extending past the U-bend portioninto the straight away portion 120. The flow path of the culture in theopen raceway pond bioreactor 100 of FIG. 2 would be counter clockwise,with the culture encountering first end 140 of the turning vane first,second end 141 of the turning vane second, and then the submergedthruster 104 when traveling through the U-bend portion 130 and into thestraight away portion 120. The arched turning vanes 103 are also shownin FIGS. 1 and 3 to be at least as tall as the center wall 102, to allowa portion of the arched turning vanes 103 to protrude from the culturevolume when operating.

An embodiment of a pair of arched turning vanes for use in the U-bendportions of an open race pond bioreactor is shown in FIG. 5 with aninner turning vane 200 and an outer turning vane 300. Both turning vanesshown in FIG. 5 are asymmetrical, but the turning vanes may also besymmetrical or a combination of symmetrical and asymmetrical. Theturning vanes may also have the same curvature radius or differentcurvature radii.

An embodiment for structurally supporting an arched turning vane isshown in the view of FIG. 6. The arched turning vane 603 is supported bya plurality of structural support members 607 fastened to the outer wall601 and the center wall 602 to stabilize the arched turning vane 603 andmaintain the boundaries of the flow path through the U-bend portiondefined by the outer wall 601, arched turning vane 603, and center wall602.

An embodiment of a large volume above ground open raceway pondbioreactor with multiple turning vanes and multiple submerged thrustersis shown in FIG. 7. The open raceway pond bioreactor 700 comprises anouter wall 701, center wall 702, arched turning vanes 703, supportstructure 707 for the arched turning vanes, submerged thrusters 704, andsupport structure 705 (horizontal), 706 (vertical) for the submergedthrusters 704. The outer wall 701 and the center wall 702 form thestraight away portions 720 and U-bend portions 730 of the bioreactor.The configuration of arched turning vanes 703, support structure 707 forthe arched turning vanes, submerged thrusters 704, and support structure705 (horizontal), 706 (vertical) for the submerged thrusters 704 is thesame for both ends of the bioreactor 700.

An embodiment of a turning vane also functioning as a heat exchangerdisposed in the U-bend portion of a raceway pond bioreactor is shown inFIG. 8. An inlet fluid conduit 810 and an outlet fluid conduit 812 arein fluid communication with the heat exchanger turning vane 803 thatcomprises an internal cavity for circulating a heat exchange fluid. Theinlet fluid conduit 810 comprises a valve 811 for controlling the flowof heat exchange fluid. The heat exchanger turning vane 803 is disposedbetween the outer wall 801 and the center wall 802 in the same manner asa conventional turning vane.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

REFERENCES

-   -   WO 2012/109375 A2, Postier et al.;    -   U.S. Pat. No. 4,005,546, Oswald;    -   U.S. Pat. No. 4,452,227, Lowrey, III;    -   U.S. Pat. No. 4,510,920, Walmet;    -   U.S. Pat. No. 4,643,830, Reid;    -   U.S. Pat. No. 6,659,044 B2, Salinas;    -   U.S. Pat. No. 6,852,225 B1, Oswald et al.;    -   U.S. Pat. No. 8,535,532 B2, Ott;    -   US 2008/0311646 A1, Cong et al.;    -   US 2011/0294196 A1, Machin;    -   US 2011/0318816 A1, Hazlebeck;    -   US 2012/0088296 A1, Vargas et al.;    -   US 2013/0095544 A1, Berlowitz et al.;    -   US 2013/0164834 A1, Licamele;    -   US 2013/0269244 A1, Jovine;    -   WO 2009/090521 A2, Brander, et al.;    -   WO 2012/166883 A1, Licamele et al.;    -   WO 2013/86626 A1 Lali;    -   U.S. Pat. No. 8,642,325 Benjauthrit et al.;    -   Xylem. Flygt compact mixers. Accessed online Mar. 13, 2014 at        hppt://www.flygt.com/en-us/Mixing/Products/Flygt-compact-mixers-4600-series/Documents/1103_Master_Lo.pdf.

What is claimed is:
 1. A mixotrophic bioreactor system, comprising: a.An open raceway pond comprising: i. An inner volume; ii. Two straightaway portions separated by a center wall and bounded by straight outerwalls and a floor; and iii. Two U-bend portions connecting the twostraight away portions to form a continuous looped flow path, andbounded by a curved outer wall and a floor; b. At least one submersiblethruster disposed within the inner volume a distance from the floor,wherein the at least one thruster is positioned at a U-bend exit andconfigured to maximize thrust in a straight line flow of an aqueousliquid culture through the straight away portions and add momentum tothe aqueous liquid culture along the looped flow path as it exits theU-bend portions of the looped flow path where some velocity of theaqueous liquid culture may be lost due to change in flow direction; c.At least one support structure coupled to at least one of the centerwall and an outer wall, the support structure suspending the at leastone submersible thruster from above at a position within the innervolume of the open raceway pond; and d. At least one organic carbondelivery device comprising an outlet positioned to deliver an organiccarbon source to the inner volume of the open raceway pond.
 2. Themixotrophic bioreactor system of claim 1, further comprising at leastone dissolved oxygen delivery device selected from group consisting ofaeration tubing, a sparger tube, a membrane lining at least part of thefloor of the raceway pond, a microbubble generator, an oxygenconcentrator, a liquid oxygen injector, an oxygen saturation cone, andventuri injection by a foam fractionation device.
 3. The mixotrophicbioreactor system of claim 2, wherein the dissolved oxygen deliverydevice is aeration tubing or a sparger tube.
 4. The mixotrophicbioreactor system of claim 3, wherein oxygen is supplied to the cultureby venturi injection.
 5. The mixotrophic bioreactor system of claim 1,wherein the at least one organic carbon delivery device comprises a pHauxostat system.
 6. The mixotrophic bioreactor system of claim 1,wherein the at least one thruster comprises multiple submersiblethrusters and the thrusters are disposed within 20% of the U-bend end ofthe straight away portions.
 7. The mixotrophic bioreactor system ofclaim 1, wherein the at least one submersible thruster comprisesmultiple submersible thrusters positioned parallel to each other and/orat spaced intervals within the inner volume of the open raceway pond. 8.The mixotrophic bioreactor system of claim 1, wherein the at least onesubmersible thruster comprises multiple submersible thrusters positionedin a staggered arrangement with relation to each other within the innervolume of the open raceway pond.
 9. The mixotrophic bioreactor system ofclaim 1, wherein the at least one submersible thruster is disposedbetween the center wall and the outer wall of at least one of thestraight away portions.
 10. The mixotrophic bioreactor system of claim1, wherein the at least one submersible thruster is oriented tocirculate a fluid medium through the continuous loop of the open racewaypond.
 11. The mixotrophic bioreactor system of claim 1, furthercomprising at least one heat exchanger.
 12. The mixotrophic bioreactorsystem of claim 11, wherein the at least one heat exchanger is disposedwithin at least one from the group consisting of the outer walls, thecenter wall, the floor, and under the floor of the open raceway pond.13. The mixotrophic bioreactor system of claim 1, further comprising acover over at least part of the open raceway pond.
 14. A mixotrophicbioreactor system, comprising: a. An open raceway pond comprising: i. Afloor; ii. Two straight away portions separated by a center wall andbounded by straight outer walls and the floor; and iii. Two U-bendportions connecting the two straight away portions to form a continuouslooped flow path, and bounded by a curved outer wall and the floor; iv.Wherein the floor is flat to create an inner volume that has aconsistent depth along the entire looped flow path; b. At least onesubmersible thruster disposed within the inner volume a distance fromthe floor, wherein the at least one thruster is positioned at a U-bendexit and configured to maximize thrust in a straight line flow of anaqueous liquid culture through the straight away portions and addmomentum to the aqueous liquid culture along the looped flow path as itexits the U-bend portions of the looped flow path where some velocity ofthe aqueous liquid culture may be lost due to change in flow direction;c. At least one support structure coupled to at least one of the centerwall and an outer wall, the support structure suspending the at leastone submersible thruster from above at a position within the innervolume of the open raceway pond; and d. At least one organic carbondelivery device comprising an outlet positioned to deliver an organiccarbon source to the inner volume of the open raceway pond.
 15. Themixotrophic bioreactor system of claim 14, further comprising at leastone dissolved oxygen delivery device selected from group consisting ofaeration tubing, a sparger tube, a membrane lining at least part of thefloor of the raceway pond, a microbubble generator, an oxygenconcentrator, a liquid oxygen injector, an oxygen saturation cone, andventuri injection by a foam fractionation device.
 16. The mixotrophicbioreactor system of claim 15, wherein the dissolved oxygen deliverydevice is aeration tubing or a sparger tube.
 17. The mixotrophicbioreactor system of claim 16, wherein oxygen is supplied to the cultureby venturi injection.
 18. The mixotrophic bioreactor system of claim 14,wherein the at least one organic carbon delivery device comprises a pHauxostat system.
 19. The mixotrophic bioreactor system of claim 14,wherein the at least one thruster comprises multiple submersiblethrusters and the thrusters are disposed within 20% of the U-bend end ofthe straight away portions.
 20. The mixotrophic bioreactor system ofclaim 14, wherein the at least one submersible thruster is oriented tocirculate a fluid medium through the continuous loop of the open racewaypond.